A direct interaction between CPF and RNA Pol II links RNA 3' end processing to transcription.

Transcription termination by RNA polymerase II (RNA Pol II) is linked to RNA 3' end processing by the cleavage and polyadenylation factor (CPF or CPSF). CPF contains endonuclease, poly(A) polymerase, and protein phosphatase activities, which cleave and polyadenylate pre-mRNAs and dephosphorylate RNA Pol II to control transcription. Exactly how the RNA 3' end processing machinery is coupled to transcription remains unclear. Here, we combine in vitro reconstitution, structural studies, and genome-wide analyses to show that yeast CPF physically and functionally interacts with RNA Pol II. Surprisingly, CPF-mediated dephosphorylation promotes the formation of an RNA Pol II stalk-to-stalk homodimer in vitro. This dimer is compatible with transcription but not with the binding of transcription elongation factors. Disruption of the dimerization interface in cells causes transcription defects, including altered RNA Pol II abundance on protein-coding genes, tRNA genes, and intergenic regions. We hypothesize that RNA Pol II dimerization may provide a mechanistic basis for the allosteric model of transcription termination.

Competition between maturation and degradation drives human snRNA 3' end quality control.

Polymerases and exonucleases act on 3' ends of nascent RNAs to promote their maturation or degradation but how the balance between these activities is controlled to dictate the fates of cellular RNAs remains poorly understood. Here, we identify a central role for the human DEDD deadenylase TOE1 in distinguishing the fates of small nuclear (sn)RNAs of the spliceosome from unstable genome-encoded snRNA variants. We found that TOE1 promotes maturation of all regular RNA polymerase II transcribed snRNAs of the major and minor spliceosomes by removing posttranscriptional oligo(A) tails, trimming 3' ends, and preventing nuclear exosome targeting. In contrast, TOE1 promotes little to no maturation of tested U1 variant snRNAs, which are instead targeted by the nuclear exosome. These observations suggest that TOE1 is positioned at the center of a 3' end quality control pathway that selectively promotes maturation and stability of regular snRNAs while leaving snRNA variants unprocessed and exposed to degradation in what could be a widespread mechanism of RNA quality control given the large number of noncoding RNAs processed by DEDD deadenylases.

The Y3** ncRNA promotes the 3' end processing of histone mRNAs.

We demonstrate that the Y3/Y3** noncoding RNAs (ncRNAs) bind to the CPSF (cleavage and polyadenylation specificity factor) and that Y3** associates with the 3' untranslated region (UTR) of histone pre-mRNAs. The depletion of Y3** impairs the 3' end processing of histone pre-mRNAs as well as the formation and protein dynamics of histone locus bodies (HLBs), the site of histone mRNA synthesis and processing. HLB morphology is also disturbed by knockdown of the CPSF but not the U7-snRNP components. In conclusion, we propose that the Y3** ncRNA promotes the 3' end processing of histone pre-mRNAs by enhancing the recruitment of the CPSF to histone pre-mRNAs at HLBs.

The host Integrator complex acts in transcription-independent maturation of herpesvirus microRNA 3' ends.

Herpesvirus saimiri (HVS) is an oncogenic γ-herpesvirus that produces microRNAs (miRNAs) by cotranscription of precursor miRNA (pre-miRNA) hairpins immediately downstream from viral small nuclear RNAs (snRNA). The host cell Integrator complex, which recognizes the snRNA 3' end processing signal (3' box), generates the 5' ends of HVS pre-miRNA hairpins. Here, we identify a novel 3' box-like sequence (miRNA 3' box) downstream from HVS pre-miRNAs that is essential for miRNA biogenesis. In vivo knockdown and rescue experiments confirmed that the 3' end processing of HVS pre-miRNAs also depends on Integrator activity. Interaction between Integrator and HVS primary miRNA (pri-miRNA) substrates that contain only the miRNA 3' box was confirmed by coimmunoprecipitation and an in situ proximity ligation assay (PLA) that we developed to localize specific transient RNA-protein interactions inside cells. Surprisingly, in contrast to snRNA 3' end processing, HVS pre-miRNA 3' end processing by Integrator can be uncoupled from transcription, enabling new approaches to study Integrator enzymology.

Deep sequencing of tRNA's 3'-termini sheds light on CCA-tail integrity and maturation.

The 3'-termini of tRNA are the point of amino acid linkage and thus crucial for their function in delivering amino acids to the ribosome and other enzymes. Therefore, to provide tRNA functionality, cells have to ensure the integrity of the 3'-terminal CCA-tail, which is generated during maturation by the 3'-trailer processing machinery and maintained by the CCA-adding enzyme. We developed a new tRNA sequencing method that is specifically tailored to assess the 3'-termini of E. coli tRNA. Intriguingly, we found a significant fraction of tRNAs with damaged CCA-tails under exponential growth conditions and, surprisingly, this fraction decreased upon transition into stationary phase. Interestingly, tRNAs bearing guanine as a discriminator base are generally unaffected by CCA-tail damage. In addition, we showed tRNA species-specific 3'-trailer processing patterns and reproduced in vitro findings on preferences of the maturation enzyme RNase T in vivo.

Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33.

Cleavage and polyadenylation specificity factor (CPSF) is the central component of the 3' processing machinery for polyadenylated mRNAs in metazoans: CPSF recognizes the polyadenylation signal AAUAAA, providing sequence specificity in both pre-mRNA cleavage and polyadenylation, and catalyzes pre-mRNA cleavage. Here we show that of the seven polypeptides that have been proposed to constitute CPSF, only four (CPSF160, CPSF30, hFip1, and WDR33) are necessary and sufficient to reconstitute a CPSF subcomplex active in AAUAAA-dependent polyadenylation, whereas CPSF100, CPSF73, and symplekin are dispensable. WDR33 is required for binding of reconstituted CPSF to AAUAAA-containing RNA and can be specifically UV cross-linked to such RNAs, as can CPSF30. Transcriptome-wide identification of WDR33 targets by photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) showed that WDR33 binds in and very close to the AAUAAA signal in vivo with high specificity. Thus, our data indicate that the large CPSF subunit participating in recognition of the polyadenylation signal is WDR33 and not CPSF160, as suggested by previous studies.

Promoter-proximal polyadenylation sites reduce transcription activity.

Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites on transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ~500 base pairs of the promoter. In contrast, promoter-proximal positioning of a pA site-independent histone gene terminator supports high transcription levels. We propose that optimal communication between a pA site-dependent gene terminator and its promoter critically depends on gene length and that short RNA polymerase II-transcribed genes use specialized termination mechanisms to maintain high transcription levels.

A triple helix stabilizes the 3' ends of long noncoding RNAs that lack poly(A) tails.

The MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) locus is misregulated in many human cancers and produces an abundant long nuclear-retained noncoding RNA. Despite being transcribed by RNA polymerase II, the 3' end of MALAT1 is produced not by canonical cleavage/polyadenylation but instead by recognition and cleavage of a tRNA-like structure by RNase P. Mature MALAT1 thus lacks a poly(A) tail yet is expressed at a level higher than many protein-coding genes in vivo. Here we show that the 3' ends of MALAT1 and the MEN ß long noncoding RNAs are protected from 3'-5' exonucleases by highly conserved triple helical structures. Surprisingly, when these structures are placed downstream from an ORF, the transcript is efficiently translated in vivo despite the lack of a poly(A) tail. The triple helix therefore also functions as a translational enhancer, and mutations in this region separate this translation activity from simple effects on RNA stability or transport. We further found that a transcript ending in a triple helix is efficiently repressed by microRNAs in vivo, arguing against a major role for the poly(A) tail in microRNA-mediated silencing. These results provide new insights into how transcripts that lack poly(A) tails are stabilized and regulated and suggest that RNA triple-helical structures likely have key regulatory functions in vivo.

C16orf57, a gene mutated in poikiloderma with neutropenia, encodes a putative phosphodiesterase responsible for the U6 snRNA 3' end modification.

C16orf57 encodes a human protein of unknown function, and mutations in the gene occur in poikiloderma with neutropenia (PN), which is a rare, autosomal recessive disease. Interestingly, mutations in C16orf57 were also observed among patients diagnosed with Rothmund-Thomson syndrome (RTS) and dyskeratosis congenita (DC), which are caused by mutations in genes involved in DNA repair and telomere maintenance. A genetic screen in Saccharomyces cerevisiae revealed that the yeast ortholog of C16orf57, USB1 (YLR132C), is essential for U6 small nuclear RNA (snRNA) biogenesis and cell viability. Usb1 depletion destabilized U6 snRNA, leading to splicing defects and cell growth defects, which was suppressed by the presence of multiple copies of the U6 snRNA gene SNR6. Moreover, Usb1 is essential for the generation of a unique feature of U6 snRNA; namely, the 3'-terminal phosphate. RNAi experiments in human cells followed by biochemical and functional analyses confirmed that, similar to yeast, C16orf57 encodes a protein involved in the 2',3'-cyclic phosphate formation at the 3' end of U6 snRNA. Advanced bioinformatics predicted that C16orf57 encodes a phosphodiesterase whose putative catalytic activity is essential for its function in vivo. Our results predict an unexpected molecular basis for PN, DC, and RTS and provide insight into U6 snRNA 3' end formation.

A snoRNA modulates mRNA 3' end processing and regulates the expression of a subset of mRNAs.

mRNA 3' end processing is an essential step in gene expression. It is well established that canonical eukaryotic pre-mRNA 3' processing is carried out within a macromolecular machinery consisting of dozens of trans-acting proteins. However, it is unknown whether RNAs play any role in this process. Unexpectedly, we found that a subset of small nucleolar RNAs (snoRNAs) are associated with the mammalian mRNA 3' processing complex. These snoRNAs primarily interact with Fip1, a component of cleavage and polyadenylation specificity factor (CPSF). We have functionally characterized one of these snoRNAs and our results demonstrated that the U/A-rich SNORD50A inhibits mRNA 3' processing by blocking the Fip1-poly(A) site (PAS) interaction. Consistently, SNORD50A depletion altered the Fip1-RNA interaction landscape and changed the alternative polyadenylation (APA) profiles and/or transcript levels of a subset of genes. Taken together, our data revealed a novel function for snoRNAs and provided the first evidence that non-coding RNAs may play an important role in regulating mRNA 3' processing.

Distinct roles of Pcf11 zinc-binding domains in pre-mRNA 3'-end processing.

New transcripts generated by RNA polymerase II (RNAPII) are generally processed in order to form mature mRNAs. Two key processing steps include a precise cleavage within the 3' end of the pre-mRNA, and the subsequent polymerization of adenosines to produce the poly(A) tail. In yeast, these two functions are performed by a large multi-subunit complex that includes the Cleavage Factor IA (CF IA). The four proteins Pcf11, Clp1, Rna14 and Rna15 constitute the yeast CF IA, and of these, Pcf11 is structurally the least characterized. Here, we provide evidence for the binding of two Zn2+ atoms to Pcf11, bound to separate zinc-binding domains located on each side of the Clp1 recognition region. Additional structural characterization of the second zinc-binding domain shows that it forms an unusual zinc finger fold. We further demonstrate that the two domains are not mandatory for CF IA assembly nor RNA polymerase II transcription termination, but are rather involved to different extents in the pre-mRNA 3'-end processing mechanism. Our data thus contribute to a more complete understanding of the architecture and function of Pcf11 and its role within the yeast CF IA complex.

Essential role for the interaction between hnRNP H/F and a G quadruplex in maintaining p53 pre-mRNA 3'-end processing and function during DNA damage.

Following DNA damage, mRNA 3'-end formation is inhibited, contributing to repression of mRNA synthesis. Here we investigated how DNA-damaged cells accomplish p53 mRNA 3'-end formation when normal mechanisms of pre-mRNA 3'-end processing regulation are inhibited. The underlying mechanism involves the interaction between a G-quadruplex structure located downstream from the p53 cleavage site and hnRNP H/F. Importantly, this interaction is critical for p53 expression and contributes to p53-mediated apoptosis. Our results uncover the existence of a specific rescue mechanism of 3'-end processing regulation allowing stress-induced p53 accumulation and function in apoptosis.

Crosstalk between mRNA 3' end processing and transcription initiation.

Transcription and mRNA maturation are interdependent events. Although stimulatory connections between these processes within the same round of transcription are well described, functional coupling between separate transcription cycles remains elusive. Comparing time-resolved transcription profiles of single-copy integrated ß-globin gene variants, we demonstrate that a polyadenylation site mutation decreases transcription initiation of the same gene. Upon depletion of the 3' end processing and transcription termination factor PCF11, endogenous genes exhibit a similar phenotype. Readthrough RNA polymerase II (RNAPII) engaged on polyadenylation site-mutated transcription units sequester the transcription initiation/elongation factors TBP, TFIIB and CDK9, leading to their depletion at the promoter. Additionally, high levels of TBP and TFIIB appear inside the gene body, and Ser2-phosphorylated RNAPII accumulates at the promoter. Our data demonstrate that 3' end formation stimulates transcription initiation and suggest that coordinated recycling of factors from a gene terminator back to the promoter is essential for sustaining continued transcription.

Cotranscriptional recruitment of the mRNA export factor Yra1 by direct interaction with the 3' end processing factor Pcf11.

We investigated recruitment of the yeast mRNA export factor Yra1 to the transcription elongation complex (TEC). Previously, the Sub2 helicase subunit of TREX was proposed to recruit Yra1. We report that Sub2 is dispensable for Yra1 recruitment, but the cleavage/polyadenylation factor, CF1A, is required. Yra1 binds directly to the Zn finger/Clp1 region of Pcf11, the pol II CTD-binding subunit of CF1A, and this interaction is conserved between their human homologs. Tethering of Pcf11 to nascent mRNA is sufficient to enhance Yra1 recruitment. Interaction with Pcf11 can therefore explain Yra1 binding to the TEC independently of Sub2. We propose that after initially binding to Pcf11, Yra1 is transferred to Sub2. Consistent with this idea, Pcf11 binds the same regions of Yra1 that also contact Sub2, indicating a mutually exclusive interaction. These results suggest a mechanism for cotranscriptional assembly of the export competent mRNP and for coordinating export with 3' end processing.

FLASH, a proapoptotic protein involved in activation of caspase-8, is essential for 3' end processing of histone pre-mRNAs.

3' end processing of histone pre-mRNA requires U7 snRNP, which binds downstream of the cleavage site and recruits the endonuclease CPSF-73. U7 snRNP contains a unique Sm ring in which the canonical SmD2 protein is replaced by Lsm11. We used the yeast two-hybrid system to identify binding partners of Lsm11 and selected the proapoptotic protein FLASH. Human FLASH interacts with Lsm11 in vitro and stimulates 3' end processing of histone pre-mRNA in mammalian nuclear extracts. We also identified the FLASH ortholog in Drosophila and demonstrate that it interacts with Lsm11 in vitro and in vivo. Drosophila FLASH localizes to histone locus bodies, and its depletion from fly cells inhibits U7-dependent processing, resulting in polyadenylation of histone mRNAs. These results demonstrate that FLASH is an essential factor required for 3' end maturation of histone mRNAs in both vertebrates and invertebrates and suggest a potential link between this process and apoptosis.

HIV-1 mRNA 3' end processing is distinctively regulated by eIF3f, CDK11, and splice factor 9G8.

A genetic screen previously identified the N-terminal 91 amino acids of the eukaryotic initiation factor 3 subunit f (N91-eIF3f) as a potent inhibitor of HIV-1 replication. Overexpression of N91-eIF3f or full-length eIF3f reduced the level of HIV-1 mRNAs in the infected cell. Here we show that N91-eIF3f and eIF3f act by specifically blocking the 3' end processing of the HIV-1 pre-mRNA both in vivo and in vitro. Furthermore, the results suggest that eIF3f mediates this restriction of HIV-1 expression through the previously unsuspected involvement of a set of factors that includes eIF3f, the SR protein 9G8, and the cyclin-dependent kinase 11 (CDK11). eIF3f affects HIV-1 3' end processing by modulating the sequence-specific recognition of the HIV-1 pre-mRNA by 9G8.

Transcription termination by nuclear RNA polymerases.

Gene transcription in the cell nucleus is a complex and highly regulated process. Transcription in eukaryotes requires three distinct RNA polymerases, each of which employs its own mechanisms for initiation, elongation, and termination. Termination mechanisms vary considerably, ranging from relatively simple to exceptionally complex. In this review, we describe the present state of knowledge on how each of the three RNA polymerases terminates and how mechanisms are conserved, or vary, from yeast to human.

tRNA 3' processing in yeast involves tRNase Z, Rex1, and Rrp6.

Mature tRNA 3' ends in the yeast Saccharomyces cerevisiae are generated by two pathways: endonucleolytic and exonucleolytic. Although two exonucleases, Rex1 and Rrp6, have been shown to be responsible for the exonucleolytic trimming, the identity of the endonuclease has been inferred from other systems but not confirmed in vivo. Here, we show that the yeast tRNA 3' endonuclease tRNase Z, Trz1, is catalyzing endonucleolytic tRNA 3' processing. The majority of analyzed tRNAs utilize both pathways, with a preference for the endonucleolytic one. However, 3'-end processing of precursors with long 3' trailers depends to a greater extent on Trz1. In addition to its function in the nucleus, Trz1 processes the 3' ends of mitochondrial tRNAs, contributing to the general RNA metabolism in this organelle.

Reversible and rapid transfer-RNA deactivation as a mechanism of translational repression in stress.

Stress-induced changes of gene expression are crucial for survival of eukaryotic cells. Regulation at the level of translation provides the necessary plasticity for immediate changes of cellular activities and protein levels. In this study, we demonstrate that exposure to oxidative stress results in a quick repression of translation by deactivation of the aminoacyl-ends of all transfer-RNA (tRNA). An oxidative-stress activated nuclease, angiogenin, cleaves first within the conserved single-stranded 3'-CCA termini of all tRNAs, thereby blocking their use in translation. This CCA deactivation is reversible and quickly repairable by the CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase]. Through this mechanism the eukaryotic cell dynamically represses and reactivates translation at low metabolic costs.

Positive and negative feedback loops in the p53 and mRNA 3' processing pathways.

Although the p53 network has been intensively studied, genetic analyses long hinted at the existence of components that remained elusive. Recent studies have shown regulation of p53 at the mRNA level mediated via both the 5' and the 3' untranslated regions and affecting the stability and translation efficiency of the p53 mRNA. Here, we provide evidence of a feedback loop between p53 and the poly(A)-specific ribonuclease (PARN), in which PARN deadenylase keeps p53 levels low in nonstress conditions by destabilizing p53 mRNA, and the UV-induced increase in p53 activates PARN deadenylase, regulating gene expression during DNA damage response in a transactivation-independent manner. This model is innovative because it provides insights into p53 function and the mechanisms behind the regulation of mRNA 3' end processing in different cellular conditions.